Location: Crops Pathology and Genetics ResearchTitle: PhOBF1, a petunia OCS element binding factor, plays an important role in antiviral RNA silencing
|SUN, DAOYANG - University Of California|
|LI, SHAOHUA - University Of California|
|NIU, LIXIN - Northwest Agricultural & Forestry University|
|REID, MICHAEL - University Of California|
|ZHANG, YANLONG - Northwest Agricultural & Forestry University|
Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2016
Publication Date: 1/4/2017
Citation: Sun, D., Li, S., Niu, L., Reid, M., Zhang, Y., Jiang, C. 2017. PhOBF1, a petunia OCS element binding factor, plays an important role in antiviral RNA silencing. Journal of Experimental Botany. 68(5):915-930. doi: 10.1093/jxb/erw490.
Interpretive Summary: Virus-induced gene silencing (VIGS), related to post transcriptional gene silencing (PTGS), is an attractively quick approach for degradation of homologous RNA molecules in plants. This RNA silencing machinery employs a number of core components, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs), to implement small interfering RNA (siRNA)-mediated gene knockdown in a sequence-specific manner. The Arabidopsis genome contains four DCLs, six RDRs, and ten AGOs, respectively contributing to the siRNA biogenesis, double-stranded RNA (dsRNA) formation, and incorporation into a RNA induced silencing complex (RISC). To date, however, how these RNA silencing components are transcriptionally regulated remains largely unknown. Salicylic acid (SA) is an endogenous phytohormone required for induction of systemic acquired resistance (SAR). Exogenous application of SA inhibits virus proliferation of CMV, TMV, and TCV in Arabidopsis, tomato, tobacco and hot pepper. To date, the research concerning SA mainly focuses on plant defense signaling against abiotic and biotic elicitors. Several lines of evidence support that a close correlation between SA-mediated viral defense and RNA silencing pathway may exist in plants. SA-deficient transgenic tobacco plants exhibit a reduced accumulation of Plum pox virus (PPV)-derived small RNAs, demonstrating that SA may serve as an enhancer of antiviral RNA silencing. The latest finding indicates that transcripts of RDR1, RDR2, DCL1, and DCL2 are up-regulated in tomato plants following Citrus exocortis viroid (CEVd), Tomato mosaic virus (ToMV) infection, and SA treatments. The basic leucine zipper (bZIP) proteins constitute a large transcription factor family in eukaryotes. In Arabidopsis, the bZIP family is classified into ten subfamilies (A-I, S), of which the subfamily S is the largest group including 17 members. AtbZIP11 belongs to subfamily S and its translation is suppressed by sucrose targeting a conserved upstream open reading frame (uORF) region in the 5´ untranslated regions of mRNA. AtbZIP11 regulates amino acid metabolism by directly interacting with asparagine synthetase 1 (ASN1) and proline dehydrogenase 2 (ProDH2). Activation of AtbZIP11 leads to reprogramming of amino acids and sugar metabolisms, including increased levels of phenylalanine, tryptophan, tyrosine, sucrose, fructose, glucose, and reduced levels of trehalose, as well as promoted expression of corresponding metabolic-associated genes. Plant growth is inhibited in Arabidopsis plant constitutively expressing AtbZIP11 via the regulatory mechanism associating with the sucrose non-fermenting-1 related protein kinase 1 (SnRK1) and trehalose 6-phosphate (T6P) signaling systems. But the roles of bZIP11 in antiviral RNA silencing still remain enigmatic. In previous studies, we obtained numerous genes up-regulated, including a large number of transcription factors, during flower development through transcriptomic analysis related to flower senescence in petunia. We have used successfully TRV-based VIGS method to assay the function of senescence-related genes in petunia floral tissues. However, when the TRV-PhCHS report system was employed to silence several candidate genes, the inoculated plants failed to show the expected white-petal phenotype of CHS-silencing in the purple-petal petunia plants. Of the candidate genes, we recently have reported an ethylene-responsive element binding factor PhERF2, which plays a critical role in TRV-induced RNA silencing and antiviral defense via transcriptional modulation of RDR2, RDR6, DCL2, and AGO2 in petunia. In this study, we reported another regulatory gene, an ocs element binding factor of bZIP transcription factors, namely PhOBF1. Simultaneous silencing of PhOBF1 and reporter genes, phytoene desaturase (PDS) or chalcone synthase (CHS), by TRV
Technical Abstract: Virus-induced gene silencing (VIGS) is a common strategy of reverse genetics for characterizing function of genes in plant. The detailed mechanism governing RNA silencing efficiency triggered by virus is largely unclear. Here, we revealed that a petunia (Petunia hybrida) ocs element binding factor, PhOBF1, one of basic-leucine zipper (bZIP) transcription factors, was up-regulated by Tobacco rattle virus (TRV) infection. Simultaneous silencing of PhOBF1 and reporter genes, phytoene desaturase (PDS) or chalcone synthase (CHS), by TRV-based VIGS led to a failure of the development of leaf photobleaching or white-corollas phenotype. PhOBF1 silencing caused down-regulation of RNA silencing-related genes, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonautes (AGOs). After inoculation with the TRV-PhPDS, PhOBF1-RNAi lines exhibited a substantially impaired PDS silencing efficiency, whereas overexpression of PhOBF1 resulted in a recovery of silencing phenotype (photobleaching) in systemic leaves. A compromised resistance to TRV and Tobacco mosaic virus (TMV) was found in PhOBF1-RNAi lines, while PhOBF1-overexpressing lines displayed an enhanced resistance to their infections. Compared with wild-type (WT) plants, PhOBF1-silenced plants accumulated lower levels of free salicylic acid (SA), salicylic acid glucoside (SAG), and phenylalanine, contrarily to higher levels of those in plants overexpressing PhOBF1. Furthermore, transcripts of a number of genes associated with the shikimate and phenylpropanoid pathways were decreased or increased in PhOBF1-RNAi or -overexpressing lines, respectively. Taken together, the data suggest that PhOBF1 regulates TRV-induced RNA silencing efficiency through modulation of RDRs, DCLs, and AGOs mediated by SA biosynthesis pathway.